The present invention relates to a double-resonance structure for DNP experiments and/or ENDOR experiments. Furthermore, it relates to a method for investigating samples by DNP and/or ENDOR.
Nuclear magnetic resonance spectroscopy (NMR spectroscopy) is one of the most important spectrographic methods for elucidating the structure and dynamics of molecules, in particular in organic chemistry and biochemistry. However, the sensitivity of NMR spectrometers reaches its limits in many applications, for example, in investigation of large biomolecules in vitro and in vivo. The lack of sensitivity can be improved to a certain extent by applying a stronger external magnetic field, but this is possible only to a limited extent and at a very high cost.
A very promising alternative for increasing the sensitivity of NMR measurements in biomolecules, for example, consists of a method known as “dynamic nuclear polarization” or “DNP method” from the abbreviation of the English term “dynamic nuclear polarization.” DNP results from the transfer of spin polarization from electrons to nuclei according to the principle known as the “Overhauser effect.” To make DNP usable in NMR spectroscopy, the electron spin polarizations must first be transferred to the nuclear spin system. To do so, the sample is excited at an electron spin resonance frequency, usually referred to as the EPR frequency, where EPR is the abbreviation for the English term “electronic paramagnetic resonance.” The EPR frequency, also known as the Larmor frequency, corresponds to the splitting of energy of electron spin energy quantum states of an atom or molecule in an external magnetic field according to the Zeeman effect, which would be degenerate in the absence of an external magnetic field. The splitting of the energy states is proportional to the strength B of the external magnetic field, and thus the value of the EPR frequency is a function of the magnetic field strength. However, the EPR frequency is always in the microwave range in applications of practical relevance. The change in polarization of the electron spin due to input of EPR microwaves is often referred graphically to as “pumping”.
The amplification of the NMR signals due to DNP is proportional to the square of the intensity of the EPR microwave field as long as the EPR transitions are not saturated. Microwave resonators in which the sample is arranged for stimulation of the EPR transitions are preferably used to obtain an EPR microwave field with the highest possible power and/or field strength.
As in EPR, nuclear magnetic resonance (NMR) is also based on transitions between quantum states of a spin in an external magnetic field, except that the energy splitting of the nuclear spins is much smaller than in EPR. NMR frequencies are typically in a two-digit megahertz range, i.e., still in the high-frequency (HF) range. The literature also uses the term “radio frequency” instead of the term “high frequency”. However, the term “high frequency” should not make us forget that these NMR frequencies are actually the lower frequencies of the system, namely lower than the aforementioned microwave frequencies. Since a high intensity of the HF field is also necessary for NMR spectroscopy, an HF resonance coil is also preferably used. So-called double-resonance structures, which have a microwave resonator for EPR transitions and an HF coil for NMR transitions, are therefore advantageous, so that the same sample may be arranged at the same time in a MW field and in an HF field with high intensities.
So-called “electron nuclear double-resonance spectroscopy” or ENDOR spectroscopy is a method related to DNP-NMR spectroscopy in terms of its conception. ENDOR spectroscopy is a special type of EPR spectroscopy, in which NMR transitions in a sample are induced by applying HF fields. To this extent, ENDOR spectroscopy is conceptually very similar to DNP-NMR spectroscopy, except that in this case pumping is performed in HF fields, and EPR spectroscopy is performed. A double-resonance structure is also used for ENDOR experiments.
A double-resonance structure is known from the article by Weis et al. (High-field DNP and ENDOR with novel multiple-frequency resonance structure, J. Magn. Reson. 140, 293-299 (1999)). This previously known double-resonance structure comprises a cylindrical microwave resonator formed from a helically coiled conductive strip. The helically coiled conductive strip forms a coil which assumes the function of the HF resonator. The cylindrical MW resonator is therefore also referred to as the helix resonator. An iris is formed in the lateral surface of the helix through which microwaves can be fed to the helix resonator. The length of the resonator may be adjusted by adjustable pistons inserted into the helix at both ends.
In the known helix resonator, a cylindrical TE011 microwave mode can be excited so that a very high microwave energy density can be achieved in the MW resonator. However, the dimensions of the helix resonator correlate with the wavelength, and if the wavelength of the microwaves drops below a millimeter in strong magnetic fields according to the EPR conditions, the small size of the helix resonator limits the sample volume that can be accommodated in the helix resonator.
In the case of liquid samples, in particular aqueous samples, there is also the problem in using the known helix resonator that far less than its entire volume can be used for accommodating the sample volume because the sample would heat up too much from the application of microwaves. The reason for the strong heating is the frequency-dependant dielectric permittivity of water under the influence of microwaves. For example, the complex dielectric permittivity of water at a microwave frequency of 260 GHz has a real part ∈′=5.6 and an imaginary part ∈″=5.8, where the dielectric losses are proportional to the imaginary part ∈″ of the permittivity. The relatively strong losses, also known as “insertion losses”, result in the fact that, on the one hand, the MW field in the sample is considerably weaker than the field outside of the sample, and on the other hand, the sample is heated strongly.
For example, if biomolecules in aqueous solutions are to be investigated, high heating of the sample is out of the question because the biomolecules might be destroyed by heating. The present inventors used an aqueous sample in a capillary with a diameter of only 0.1 mm on a trial basis and found that the temperature of the sample was raised by 90° C. under the influence of microwaves. Even at a capillary diameter of only 0.05 mm, there was still a heating of 17° C. This means that the sample volume must always be kept relatively small, so that the filling factor
  η  =                    V        s            ⁢                        〈                      B            HF            2                    〉                s                            V        struk            ⁢                        〈                      B            HF            2                    〉                struk            is relatively small here, leading to a reduced NMR sensitivity. Herein, Vs is the volume of the sample, <B2HF> is the average value of the magnetic field strength <B2HF>s in the area of the sample, Vstruk is the volume of the structure and <B2HF>struk is the average value of the magnetic field strength BHF of the field in the area of the structure. If the MW power is reduced to prevent excessive heating of the sample, this leads to a weakening of the DNP and thus in turn to a negative effect on the NMR sensitivity.
Another double-resonance structure, a so-called cavity resonator for ENDOR is described in JP2005-121409. The resonator has an HF coil wound around the sample, which is in turn arranged in an MW cavity. This structure is suitable for ENDOR spectroscopy but not for DNP applications because the HF coil leads to a disturbance in the distribution of the electrical MW field over the sample volume, which leads to disadvantageous heating of the sample.
The object of the present invention is to provide a double-resonance structure and a method for investigating specimens by means of DNP-NMR and/or ENDOR, which will allow an increased measurement sensitivity.